Methods and Compositions for Selective Regulation of Protein Expression

ABSTRACT

The invention provides methods and compositions for selectively suppressing the expression of a recombinant protein in a male reproductive tissue of a transgenic plant. The invention also provides methods and compositions for inducing male sterility in a transgenic plant. Plants, plant cells, plant parts, seeds, and commodity products including such compositions are aspects of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalPatent Application 61/504,102, which was filed on Jul. 1, 2011, which isincorporated herein by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

The sequence listing contained in the file named “MONS294US.txt”, whichis 40.5 kilobytes (size as measured in Microsoft Windows(D) and wascreated on Jun. 15, 2012, is filed herewith by electronic submission andis incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates generally to the fields of agriculture, plantbreeding, and molecular biology. More specifically, the inventionrelates to methods and compositions for selectively suppressingrecombinant protein expression in the male reproductive tissue oftransgenic plants and uses thereof.

BACKGROUND OF THE INVENTION

Hybrid seed, that is, seed produced by hybridization orcross-fertilization of closely related plants, can be grown into progenyhybrid plants possessing a desirable combination of traits not possessedby either parent plant. Hybrid plants can display superior agronomiccharacteristics, including improvement of plant size, yield, nutritionalcomposition, disease resistance, herbicide tolerance, stress tolerance,climatic adaptation, and other desirable traits. Efficient hybrid seedproduction requires that a plant's own pollen not be permitted toself-fertilize the plant.

In hybrid seed production, pollen production and/or shed may beprevented in a female parent plant in order to facilitatecross-pollination of the female rather than self-pollination. Suchprevention may be achieved by, for example, manual removal of thepollen-containing structures (e.g., manual or mechanical detasseling incorn), use of a genetic means of pollination control (e.g., cytoplasmicmale sterile, nuclear male sterile), and/or use of a chemical agent.

SUMMARY OF THE INVENTION

The invention relates generally to methods of selectively suppressingrecombinant protein expression in the male reproductive tissue oftransgenic plants, recombinant DNA constructs useful in such methods, aswell as transgenic plants, cells, and seeds containing such recombinantDNA constructs. The recombinant DNA constructs and the transgenicplants, cells, and seeds containing such constructs provide a greatlyimproved way to use herbicides for inducing male sterility in transgenicplants for the production of hybrid seed.

In one aspect, the invention provides a recombinant DNA construct thatincludes a protein-coding sequence encoding a recombinant protein and amale tissue-specific siRNA (mts-siRNA) element operably linked to theprotein-coding sequence. In one embodiment, the mts-siRNA element isincluded within the 3′ untranslated region of the protein-codingsequence. In another embodiment, the mts-siRNA element is locatedbetween the protein-coding sequence and a polyadenylation sequence whichis part of a 3′ untranslated region. In another embodiment, themts-siRNA element includes at least one mts-siRNA sequence. In anotherembodiment, the mts-siRNA element includes at least one mts-siRNAsequence selected from the group consisting of SEQ ID NO: 1-56 and105-149. In another embodiment, the mts-siRNA element is selected fromthe group consisting of SEQ ID NO: 57-94 and 96-104. In anotherembodiment, the expression of the recombinant protein in a transgenicplant confers at least vegetative herbicide tolerance to the plant. Inanother embodiment, the recombinant protein is a glyphosate-tolerantEPSPS.

Another aspect of the invention provides a method of making arecombinant DNA construct including identifying an mts-siRNA elementincluding at least one mts-siRNA sequence and operably linking themts-siRNA element to a protein-coding sequence, for instance a DNAsequence encoding a recombinant protein. In one embodiment, themts-siRNA element includes at least one mts-siRNA sequence selected fromthe group consisting of SEQ ID NO: 1-56 and 105-149or is at least onemts-siRNA element selected from the group consisting of SEQ ID NO: 57-94and 96-104. In another embodiment, the mts-siRNA element istassel-specific.

In a further aspect, the invention provides a transgenic plant includinga recombinant DNA construct of the invention, as well as a seed, cell,or part of the transgenic plant. In one embodiment, the plant is amonocotyledonous plant. In another embodiment, the plant is a maize (Zeamays) plant.

In a further aspect, the invention also provides a method of selectivelysuppressing the expression of a recombinant protein in a malereproductive tissue of a transgenic plant by expressing in thetransgenic plant a recombinant DNA construct that includes aprotein-coding sequence operably linked to a DNA sequence including anmts-siRNA element. In one embodiment, the mts-siRNA element includes atleast one mts-siRNA sequence. In another embodiment, the malereproductive tissue is a tassel of a maize plant. In another embodiment,the mts-siRNA element includes at least one mts-siRNA sequence selectedfrom the group consisting of SEQ ID NO: 1-56 and 105-149. In anotherembodiment, the mts-siRNA element is at least one element selected fromthe group consisting of SEQ ID NO: 57-94 and 96-104. In anotherembodiment, the expression of the recombinant protein in a transgenicplant confers at least vegetative herbicide tolerance to the plant. Inanother embodiment, the recombinant protein is a glyphosate-tolerantEPSPS.

The invention also provides a method of inducing male sterility in atransgenic plant, including the step of applying herbicide to atransgenic plant that has in its genome a recombinant DNA constructcomprising a protein-coding sequence operably linked to a DNA sequenceincluding an mts-siRNA element that confers at least vegetativeherbicide tolerance to the transgenic plant, wherein the herbicide isapplied during the development of the male reproductive tissue of thetransgenic plant thereby inducing male-sterility in the transgenicplant. In one embodiment, the transgenic plant is a maize plant. Inanother embodiment, the herbicide application prevents at least pollenshed or anther extrusion in the treated transgenic plant. In anotherembodiment, the development stage of the male reproductive tissue duringwhich herbicide is applied is a stage selected from the group consistingof the V4, V5, V6, V7, V8, V9, V10, V11, V12, V13, and V14 stage ofmaize plant development. In another embodiment, the herbicide isselected from the group consisting of acetyl coenzyme A carboxylase(ACCase) inhibitors, acetolactate synthase (ALS) inhibitors, photosystemII (PSII) inhibitors, protoporphyrinogen oxidase (PPO) inhibitors,4-hydroxyphenyl dioxygenase (HPPD) inhibitors, 5-enolypyruvyl shikimate3-phosphate synthase (EPSPS) inhibitors, glutamine synthetase (GS)inhibitors, and synthetic auxins. In another embodiment, the herbicideis glyphosate and the recombinant protein is a glyphosate-tolerantEPSPS.

The invention also provides a method of producing hybrid seed includingapplying an effective amount of an herbicide to a transgenic plantincluding in its genome a recombinant DNA construct comprising aprotein-coding sequence operably linked to a DNA sequence including anmts-siRNA element, wherein the herbicide is applied during thedevelopment of the male reproductive tissue of the transgenic plantthereby inducing male sterility in the transgenic plant; fertilizing thetransgenic plant with pollen from a second plant; and harvesting hybridseed from the transgenic plant. In one embodiment, the transgenic plantis maize. An effective amount of an herbicide is a dose of herbicidesufficient to render a transgenic plant comprising a recombinant DNAconstruct of the invention male sterile (an effective dose). In anotherembodiment, the herbicide is glyphosate and the recombinant protein is aglyphosate-tolerant EPSPS. In another embodiment, the glyphosate isapplied during the development at an effective dose of about 0.125pounds acid equivalent per acre to about 8 pounds acid equivalent peracre. Other specific embodiments of the invention are disclosed in thefollowing detailed description. Throughout this specification and theclaims, unless the context requires otherwise, the word “comprise” andits variations, such as “comprises” and “comprising”, will be understoodto imply the inclusion of a stated integer, element, or step or group ofintegers, elements, or steps, but not the exclusion of any otherinteger, element, or step or group of integers, elements, or steps.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts mapping of mts-siRNA sequences on an mts-siRNA element(SEQ ID NO: 85), as described in Example 1. The X-axis from left toright represents the orientation of the mts-siRNA element with the topstrand represented in the top half of the chart and the bottom strandrepresented in the bottom half of the chart; the nucleotide positionfrom 5′ to 3′ orientation is shown from left to right on the top andfrom right to left on the bottom. The mts-siRNA sequences are shown intheir relative alignment positions. The Y-axis represents the relativeabundance of the mts-siRNA expressed in tassel tissue as transcripts perquarter million sequences (tpq). The mts-siRNA that were highlyrepresented in the library are circled.

FIG. 2 depicts Northern blot analysis to measure tassel-specific sRNAexpression, as described in Example 2.

FIG. 3 depicts mapping of mts-siRNA sequences on an mts-siRNA element(SEQ ID NO: 87), as described in Examples 2 and 8. The X-axis from leftto right represents the orientation of the mts-siRNA element with thetop strand represented in the top half of the chart and the bottomstrand represented in the bottom half of the chart; the nucleotideposition from 5′ to 3′ orientation is shown from left to right on thetop and from right to left on the bottom. The mts-siRNA sequences areshown in their relative alignment positions. The three mts-siRNAsequences used to design three specific probes (sR648011 (SEQ ID NO: 8),sR1372590 (SEQ ID NO: 26), and sR410590 (SEQ ID NO: 33)) are indicated.

FIG. 4 depicts Northern blot analysis of tassel maturity temporalexpression of an mts-siRNA element (SEQ ID NO: 87) using RNA fromdifferent inbred germplasm, as described in Example 2.

FIG. 5 depicts in situ localization of siRNA expression in mature antherusing antisense (left panel) or sense (right panel) probes for anmts-siRNA sequence (sR648011, SEQ ID NO: 8), as described in Example 2

FIG. 6 depicts CP4-EPSPS protein localization in anthers from unsprayedplants transgenic for construct 3 (FIG. 6A) or construct 4 (FIG. 6B), asdescribed in Example 4. Construct 3 transgenic maize plants contain aCP4-EPSPS/mts-siRNA element expression cassette. Construct 4 plants area control.

FIG. 7 depicts transgenic maize plants generated from constructscontaining a CP4-EPSPS/mts-siRNA element expression cassette, which werevegetatively tolerant to glyphosate and had induced male-sterility withlate application of glyphosate, as described in Example 7. FIG. 7A showsglyphosate-sprayed and unsprayed transgenic maize plants. FIG. 7B showstassels from unsprayed transgenic plants, and FIG. 7C shows pollengrains from unsprayed transgenic plants. FIG. 7D shows tassels fromsprayed transgenic plants, and FIG. 7E shows pollen grains from sprayedtransgenic plants.

FIG. 8 depicts data for one year of field trials measuring MaleFertility Rating (MFR) following late glyphosate spray. FIG. 8A showsaverage MFR produced under three different glyphosate spray treatmentregimens (Trt 1, Trt 2, and Trt 3) for NK603 (CP4-EPSPS transgenicmaize), MON87427 (CP4-EPSPS transgenic maize with glyphosate-induciblemale-sterility), and two events from construct 3, as described inExample 5; the dashed line indicates the industry standard formale-sterility, MFR 2. FIG. 8B depicts a tassel from a plant treatedwith a weed-only spray treatment. FIG. 8C depicts a tassel from a planttreated with a late glyphosate spray treatment for inducingmale-sterility.

FIG. 9 depicts field trial results measuring the number of plants perplot with male-sterility measured by anther extrusion through S90, atS90+3, and at S90+6 under two different glyphosate spray treatmentregimens (Trt 2 and Trt 3) for NK603 (CP4-EPSPS transgenic maize),MON87427 (CP4-EPSPS transgenic maize with glyphosate-induciblemale-sterility), and four events from construct 3, as described inExample 5.

FIG. 10 depicts results of pollen viability studies as described inExample 5. FIGS. 10A and 10B show an example of late breaking antherextrusion in tassel from a sterility sprayed construct 3 event. The boxin FIG. 10A is the portion magnified in FIG. 10B. An example of latebreaking anther extrusion is circled in FIG. 10B. Alexander staining ofpollen from sterility-sprayed, late breaking extruded anther of sprayedconstruct 3 events shows only non-viable pollen (translucent light blue,irregular shape pollen grains) (FIG. 10C). Pollen from non-sprayedconstruct 3 anthers was fully viable and appears opaque, dark purple andspherical with Alexander stain (FIG. 10D).

FIG. 11 depicts results of field trial testing of NK603 plants andconstruct 3 events for inbred grain yield and male fertility, asdescribed in Example 6. Inbred yield was measured as bushels/acre(Bu/acre) and induced male-sterility was measured as Male FertilityRating (MFR). The horizontal bar indicates the industry standard formale-sterility, MFR 2. Trt 1, Trt 2, and Trt 3 refer to treatmentregimens 1, 2, and 3.

FIG. 12 depicts results of field trial testing of a non-transgenicfemale inbred (Null), line MON87427, and three events from construct 3,all in the same genetic background, which were cross pollinated with amale MON810/MON88017 tester to generate F1 hybrid seed. Hybrid grainyield was measured as bushels/acre (Bu/acre). Trt 1, Trt 2, and Trt 3refer to treatment regimens 1, 2, and 3.

FIG. 13 depicts pollen grain analysis from F1 hybrid plants, asdescribed in Example 7. The panels show Alexander staining results ofpollen from three different F1 hybrid crosses: non-transgenic female xMON88017 male; MON87427 female x MON88017 male; and construct 3 eventfemale x MON88017 male. Tassel fertility was functionally restored in F1hybrids produced from construct 3 event plants using MON88017 pollen.

FIG. 14 depicts schematic drawings of embodiments of recombinant DNAconstructs (shown in 5′ to 3′ direction from left to right) including(top) a protein-coding sequence (e.g., DNA encoding aglyphosate-resistant EPSPS) operably linked to a DNA sequence comprisingan mts-siRNA element (e.g., one or more selected from the groupconsisting of SEQ ID NO: 57-94 and 96-104) (top). In a non-limitingspecific embodiment (bottom) the recombinant DNA construct includes apromoter operably linked to, in order, an intron, a transit peptide,CP4-EPSPS encoded by SEQ ID NO: 95, an mts-siRNA element (SEQ ID NO:81), and a 3′UTR.

DETAILED DESCRIPTION OF THE INVENTION Recombinant DNA Constructs

The invention provides compositions and methods for selectivelysuppressing recombinant protein expression in a male reproductive tissueof a transgenic plant and uses thereof. In one aspect, the inventionprovides a recombinant DNA construct that includes a protein-codingsequence operably linked to a DNA sequence including an mts-siRNAelement, i.e. a chimeric transgene including a protein-coding sequenceencoding the recombinant protein and at least one mts-siRNA elementoperably linked to the protein-coding sequence. In one embodiment, suchrecombinant DNA constructs are useful for selectively suppressing theexpression of a recombinant protein in a male reproductive tissue of atransgenic plant. In one aspect, the invention provides a recombinantDNA molecule comprising the recombinant DNA construct and methods of usethereof. Nucleic acid sequences can be provided as DNA or as RNA, asspecified; disclosure of one necessarily defines the other, as is knownto one of ordinary skill in the art. Furthermore, disclosure of a givennucleic acid sequence necessarily defines the exact complement of thatsequence, as is known to one of ordinary skill in the art.

A “male tissue-specific siRNA” or “mts-siRNA” is a small RNA (sRNA) ofabout 18 to about 26 nucleotides (e.g., 18, 19, 20, 21, 22, 23, 24, 25or 26 nucleotides) enriched or specifically expressed in the malereproductive tissue(s) (e.g., male inflorescence) of a plant, i.e.,having an male tissue-specific expression pattern. Male tissue-specificsiRNA are naturally occurring in plants and can be detected usingtechniques known in the art, such as low molecular weight northernanalysis. A DNA sequence that is complementary to an mts-siRNA isreferred to herein as an “mts-siRNA sequence”. Examples of mts-siRNAsequences for endogenous plant mts-siRNA are provided as SEQ ID NO: 1-56and 105-149. In an embodiment, an mts-siRNA sequence is the exact DNAcomplement (with no mismatches) to a given mts-siRNA. In otherembodiments, an mts-siRNA sequence varies by 1-3 nucleotide mismatchescompared to a given mts-siRNA and nonetheless has sufficientcomplementarity to bind or hybridize, e.g., under typical physiologicalconditions, to that mts-siRNA. “Complementarity” refers to thecapability of nucleotides on one polynucleotide strand to base-pair withnucleotides on another polynucleotide strand according to the standardWatson-Crick complementarity rules (i e., guanine pairs with cytosine(G:C) and adenine pairs with either thymine (A:T) or uracil (A:U); it ispossible for intra-strand hybridization to occur between two or morecomplementary regions of a single polynucleotide. When included in arecombinant DNA construct as described herein, an mts-siRNA is capableof RNAi-mediated suppression or disruption of the expression of a geneand/or protein.

At least one, at least two, at least three, or more than three mts-siRNAsequences can be clustered together or even overlap within a single DNAmolecule. Such a DNA molecule is referred to herein as a “maletissue-specific siRNA element” or “mts-siRNA element” and is defined asincluding at least one, at least two, at least three, or more than threemts-siRNA sequences within an about 500 nucleotide sequence window. Anmts-siRNA element can be any length, such as about 20 nucleotides (nt),about 25 nt, about 30 nt, about 40 nt, about 50 nt, about 60 nt, about70 nt, about 80 nt, about 100 nt, about 150 nt, about 200 nt, about 250nt, about 300 nt, about 350 nt, about 400 nt, about 450 nt, about 500nt, about 550 nt, or about 600 nt.

A recombinant DNA construct of the invention is a DNA molecule includingat least a protein-coding sequence operably linked to a DNA sequenceincluding an mts-siRNA element. The term “recombinant” refers to amolecule or a cell or organism that man-made through genetic engineeringand as such is the product of human activity and would not otherwisenormally occur in nature. As used herein, a recombinant DNA construct isa recombinant DNA molecule including two or more heterologous DNAsequences. The term “heterologous” refers to the relationship betweentwo or more nucleic acid or protein sequences that are derived fromdifferent sources (e.g., from different locations in a genome, or fromdifferent species). In one example, a promoter and a protein-coding DNAsequence are heterologous with respect to each other if the promoter isnot the native promoter of the protein-coding DNA sequence. In anotherexample, a protein-coding sequence is heterologous with respect to anmts-siRNA element if such a combination is not normally found in nature,such as a plant mts-siRNA element operably linked to a gene forherbicide tolerance, such as CP4-EPSPS. In addition, a particularsequence can be “heterologous” with respect to a cell or organism intowhich it is introduced (i.e., a sequence that does not naturally occurin that particular cell or organism).

The term “operably linked” refers to two polynucleotide molecules linkedin manner so that one can affect the expression of the other. Forexample, a first polynucleotide molecule is operably linked with asecond polynucleotide molecule where the polynucleotide molecules are soarranged that the first polynucleotide molecule can affect theexpression of the second polynucleotide molecule. The two polynucleotidemolecules can be part of a single contiguous polynucleotide molecule andcan be adjacent or separated. For example, an mts-siRNA element isoperably linked to a protein-coding sequence if, after transcription inmale reproductive tissue cell, the presence of the mts-siRNA elementresults in the suppression of recombinant protein expression in thecell. Operable linkage of the protein-coding sequence and the mts-siRNAelement can be achieved, for example, through incorporation of anmts-siRNA element adjacent to the protein-coding sequence (such aslocated 5′ or 3′ to the protein-coding sequence, but not necessarily incontiguous linkage), in or adjacent to an untranslated region (UTR) ofthe recombinant DNA construct (such as located in or next to the 5′ UTRor the 3′ UTR), and/or after the protein-coding sequence and before thepolyadenylation signal. In one embodiment, one or more mts-siRNAelements are located between the protein-coding sequence and thepolyadenylation sequence, i.e., 3′ to and adjacent to the protein-codingsequence. In another embodiment, one or more mts-siRNA elements arelocated between the stop codon of the protein-coding sequence and thepolyadenylation sequence. In another embodiment, one or more mts-siRNAelements are located within the 3′ UTR sequence adjacent to theprotein-coding sequence.

The DNA sequence of the mts-siRNA element can be varied by usingdifferent combinations and locations of individual mts-siRNA sequencesand/or by incorporating 1-3 nucleotide mismatches in an mts-siRNAelement (relative to a given mts-siRNA sequence). Examples of mts-siRNAelements are provided herein as SEQ ID NO: 57-94 and 96-104 and in theworking Examples. An mts-siRNA element can function in either direction,i.e., it is non-directional, and as such can be used in either the 5′ to3′ orientation or in the 3′ to 5′ orientation in a recombinant DNAconstruct.

Mts-siRNA elements, mts-siRNA sequences, and mts-siRNAs can beidentified by methods known to those skilled in the art, for examplethrough bioinformatic analysis of plant sRNA and cDNA libraries. Anexample of such an identification method is provided in the Examplesbelow. In particular, mts-siRNA and mts-siRNA sequences can beidentified from sRNA libraries. The identified mts-siRNA sequences canbe compared to cDNA and/or genomic sequence collections to identifymts-siRNA elements (i.e., regions of DNA including at least one, atleast two, at least three, or more than three mts-siRNA sequences withina 500 nucleotide sequence window), which are useful for developingrecombinant DNA constructs as described herein.

In some embodiments, these mts-siRNA elements are synthesized ormodified in vitro to contain more, fewer, or different mts-siRNAsequences and/or to rearrange the relative position of one or moremts-siRNA sequence(s), where such a modification is beneficial inincreasing or decreasing the effect of the mts-siRNA element. Methodsfor synthesizing or for in vitro modification of an mts-siRNA elementand determining the optimal variation for the desired level ofsuppression are known by those of skill in the art. Chimeric mts-siRNAelements can also be designed using methods known to those of skill inthe art, such as by inserting additional desired mts-siRNA sequencesinternally in an mts-siRNA element or by linking additional mts-siRNAsequences 5′ or 3′ to an mts-siRNA element. Non-limiting embodiments ofa chimeric mts-siRNA element include mts-siRNA elements having about 80nt, about 100 nt, about 150 nt, about 200 nt, about 250 nt, or about 300nt of SEQ ID NO: 86; about 80 nt, about 100 nt, about 150 nt, about 200nt, about 250 nt, or about 300 nt of SEQ ID NO: 87; and/or about 80 nt,about 100 nt, about 150 nt, about 200 nt, about 250 nt, about 300 nt,about 350 nt, about 400 nt, about 450 nt, about 500 nt, or about 550 ntof SEQ ID NO: 85. Additional embodiments are provided in the workingExamples.

The recombinant DNA construct can be used to selectively suppressexpression of the recombinant protein in male reproductive tissues of atransgenic plant expressing the construct, i.e., resulting in expressionin at least vegetative tissues but not in male reproductive tissues. Asused herein, “expression of a recombinant protein” refers to theproduction of a recombinant protein from a protein-coding sequence andthe resulting transcript (mRNA) in a cell. As used herein the term“suppressing” means reducing; for example, suppressing the expression ofa recombinant protein means reducing the level of recombinant proteinproduced in a cell, e.g., through RNAi-mediated post-transcriptionalgene suppression.

Selective suppression of recombinant protein as used herein refers to areduction of recombinant protein production in a cell or tissue ascompared to a reference cell or tissue by at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,or at least about 99%. A reference cell or tissue can be, e.g., avegetative cell or tissue from the same or a similar transgenic plantexpressing the recombinant protein, or e.g., a vegetative cell or tissuefrom a transgenic plant having a similar transgene for expressing therecombinant protein but lacking the mts-siRNA element. Suppression ofprotein expression can be determined using any technique known to oneskilled in the art, such as by directly measuring protein accumulationin a cell or tissue sample using a technique such as ELISA or westernblot analysis, by measuring enzymatic activity of the protein, or byphenotypically determining protein expression. In one embodiment,selective suppression of recombinant protein refers to sufficientreduction in expression of a recombinant protein capable of conferringherbicide tolerance in the male tissue of a transgenic plant, resultingin a detectable phenotype of altered male fertility in a transgenicplant to which herbicide was applied as a sterility spray. The detectionof altered male fertility in such a transgenic plant would thereforeindicate the selective suppression of the recombinant protein.

As used herein, the term “protein-coding sequence” refers to apolynucleotide molecule having a nucleotide sequence that encodes apolypeptide or protein sequence. i.e., a polynucleotide sequenceencoding a recombinant protein. Depending upon conditions, thenucleotide sequence may or may not be actually translated into apolypeptide molecule in a cell. The boundaries of a protein-codingsequence are commonly delineated by a translation start codon at the5′-terminus and a translation stop codon at the 3′-terminus. Aprotein-coding sequence of the invention includes, but is not limitedto, a protein-coding sequence that provides a desirable characteristicassociated with plant morphology, physiology, growth and development,yield, nutritional enhancement, disease or pest resistance, herbicidetolerance, or environmental or chemical tolerance. In one embodiment, aprotein-coding sequence of the invention encodes a recombinant proteinthat when expressed in a transgenic plant confers herbicide tolerance atleast in a cell and/or tissue where the expressed protein occurs;selective suppression of the herbicide tolerance protein in malereproductive tissue of the transgenic plant in conjunction with timelyapplication of the herbicide results in at least reduced male fertilityor in male sterility. Such inducible male-sterility combined withvegetative herbicide tolerance can be used to increase the efficiencywith which hybrid seed is produced, for example by eliminating orreducing the need to physically emasculate the maize plant used as afemale in a given cross during hybrid seed production.Herbicide-inducible male-sterility systems have been described, forinstance in U.S. Pat. No.6,762,344 and U.S. Patent Publication2011/0126310. Examples of herbicides useful in practicing the inventioninclude, but are not limited to, acetyl coenzyme A carboxylase (ACCase)inhibitors (e.g., fops and dims), acetolactate synthase (ALS) inhibitors(e.g., sulfonylureas (SUs) and imidazolinones (IMIs)), photosystem II(PSII) inhibitors (e.g., traiazines and phenyl ethers),protoporphyrinogen oxidase (PPO) inhibitors (e.g., flumioxazsin andfomesafen), 4-hydroxyphenyl pyruvate dioxygenase (HPPD) inhibitors(e.g., isoxaflutole and triketones such as mesotrione), 5-enolypyruvylshikimate 3-phosphate synthase (EPSPS) inhibitors (e.g., glyphosate),glutamine synthetase (GS) inhibitors (e.g., glufosinate andphosphinothricin), synthetic auxins (e.g., 2,4-D and dicamba). Examplesof protein-coding sequences and/or recombinant proteins for use inpracticing the invention include but are not limited to genes encodingrecombinant proteins conferring tolerance to HPPD inhibitors (such asherbicide-insensitive HPPD), genes encoding recombinant proteinsconferring tolerance to glufosinate (such as pat and bar), genesencoding recombinant proteins conferring tolerance to glyphosate (suchas the glyphosate-tolerant EPSPS known as CP4-EPSPS, provided herein asSEQ ID NO: 95), and genes encoding recombinant proteins conferringtolerance to dicamba (such as dicamba monooxygenase (DMO)).

Recombinant DNA constructs of the invention are made by techniques knownin the art and in various embodiments are included in planttransformation vectors, plasmids, or plastid DNA. Such recombinant DNAconstructs are useful for producing transgenic plants and/or cells andas such can be also contained in the genomic DNA of a transgenic plant,seed, cell, or plant part. This invention therefore includes embodimentswherein the recombinant DNA construct is located within a planttransformation vector, or on a biolistic particle for transforming aplant cell, or within a chromosome or plastid of a transgenic plantcell, or within a transgenic cell, transgenic plant tissue, transgenicplant seed, transgenic pollen grain, or a transgenic or partiallytransgenic (e.g., a grafted) plant. A vector is any DNA molecule thatmay be used for the purpose of plant transformation, i.e., theintroduction of DNA into a cell. Recombinant DNA constructs of theinvention can, for example, be inserted into a plant transformationvector and used for plant transformation to produce transgenic plants,seeds, and cells. Methods for constructing plant transformation vectorsare well known in the art. Plant transformation vectors of the inventiongenerally include, but are not limited to: a suitable promoter for theexpression of an operably linked DNA, an operably linked recombinant DNAconstruct, and a polyadenylation signal (which may be included in a3′UTR sequence). Promoters useful in practicing the invention includethose that function in a plant for expression of an operably linkedpolynucleotide. Such promoters are varied and well known in the art andinclude those that are inducible, viral, synthetic, constitutive,temporally regulated, spatially regulated, and/or spatio-temporallyregulated. Additional optional components include, but are not limitedto, one or more of the following elements: 5′ UTR, enhancer, cis-actingelement, intron, signal sequence, transit peptide sequence, and one ormore selectable marker genes. In one embodiment, a plant transformationvector comprises a recombinant DNA construct.

The recombinant DNA constructs and plant transformation vectors of thisinvention are made by any method suitable to the intended application,taking into account, for example, the type of expression desired, theprotein-coding sequence (and thus herbicide tolerance) desired, andconvenience of use in the plant in which the recombinant DNA constructis to be expressed. General methods useful for manipulating DNAmolecules for making and using recombinant DNA constructs and planttransformation vectors are well known in the art and described in detailin, for example, handbooks and laboratory manuals including Sambrook andRussell, “Molecular Cloning: A Laboratory Manual” (third edition), ColdSpring Harbor Laboratory Press, NY, 2001. The recombinant DNA constructsof the invention can be modified by methods known in the art, eithercompletely or in part, e.g., for increased convenience of DNAmanipulation (such as restriction enzyme recognition sites orrecombination-based cloning sites), or for including plant-preferredsequences (such as plant-codon usage or Kozak consensus sequences), orto include sequences useful for recombinant DNA construct design (suchas spacer or linker sequences). In certain embodiments, the DNA sequenceof the recombinant DNA construct includes a DNA sequence that has beencodon-optimized for the plant in which the recombinant DNA construct isto be expressed. For example, a recombinant DNA construct to beexpressed in a plant can have all or parts of its sequencecodon-optimized for expression in a plant by methods known in the art.The recombinant DNA constructs of the invention can be stacked withother recombinant DNA for imparting additional traits (e.g., in the caseof transformed plants, traits including herbicide resistance, pestresistance, cold germination tolerance, water deficit tolerance) forexample, by expressing or suppressing other genes.

Transgenic Plant Cells and Transgenic Plants

An aspect of the invention includes transgenic plant cells, transgenicplant tissues, and transgenic plants or seeds which include arecombinant DNA construct of the invention. A further aspect of theinvention includes artificial or recombinant plant chromosomes whichinclude a recombinant DNA construct of the invention. Suitable methodsfor transformation of host plant cells for use with the currentinvention include virtually any method by which DNA can be introducedinto a cell (e.g., where a recombinant DNA construct is stablyintegrated into a plant chromosome) and are well known in the art. Anexemplary and widely utilized method for introducing a recombinant DNAconstruct into plants is the Agrobacterium transformation system, whichis well known to those of skill in the art. Transgenic plants can beregenerated from a transformed plant cell by the methods of plant cellculture. A transgenic plant homozygous with respect to a transgene canbe obtained by sexually mating (selfing) an independent segreganttransgenic plant that contains a single exogenous gene sequence toitself, for example an F0 plant, to produce F1 seed. One fourth of theF1 seed produced will be homozygous with respect to the transgene.Plants grown from germinating F1 seed can be tested for heterozygosity,typically using a SNP assay or a thermal amplification assay that allowsfor the distinction between heterozygotes and homozygotes (i.e., azygosity assay).

The invention provides a transgenic plant having in its genome arecombinant DNA construct of the invention, including, withoutlimitation, alfalfa, cotton, maize, canola, rice, soybean, and wheat,among others. The invention also provides transgenic plant cells, plantparts, and progeny of such a transgenic plant. As used herein “progeny”includes any plant, seed, plant cell, and/or plant part produced from orregenerated from a plant, seed, plant cell, and/or plant part thatincluded a recombinant DNA construct of the invention. Transgenicplants, cells, parts, and seeds produced from such plants can behomozygous or heterozygous for the recombinant DNA construct of theinvention.

Further included in this invention are embodiments wherein therecombinant DNA construct is in a commodity product produced from atransgenic plant, seed, or plant part of this invention; such commodityproducts include, but are not limited to harvested parts of a plant,crushed or whole grains or seeds of a plant, or any food or non-foodproduct comprising the recombinant DNA construct of this invention.

Methods of inducing male-sterility in Transgenic Plants and of ProducingHybrid Seed

Another aspect of the invention includes a method of inducingmale-sterility in a transgenic plant including applying an effectiveamount of an herbicide to a transgenic plant including a recombinant DNAconstruct that includes a protein-coding sequence encoding a recombinantprotein that confers herbicide tolerance to the transgenic plantoperably linked to a DNA sequence including an mts-siRNA element thatconfers at least vegetative herbicide tolerance to the transgenic plant,wherein the herbicide application is carried out during the developmentof the male reproductive tissue of the transgenic plant thereby inducingmale-sterility in the transgenic plant.

In one embodiment, the transgenic plant is a maize plant. In oneembodiment, the herbicide application prevents at least pollen shed oranther extrusion. In one embodiment, the development of the malereproductive tissue is a stage selected from the group consisting of theV4, V5, V6, V7, V8, V9, V10, V11, V12, V13, and V14 stage of maize plantdevelopment.

In one embodiment, the herbicide is selected from the group consistingof acetyl coenzyme A carboxylase (ACCase), acetolactate synthase (ALS)inhibitors, photosystem II (PSII) inhibitors, protoporphyrinogen oxidase(PPO) inhibitors, 4-hydroxyphenyl pyruvate dioxygenase (HPPD)inhibitors, 5-enolypyruvyl shikimate 3-phosphate synthase (EPSPS)inhibitors, glutamine synthetase (GS) inhibitors, and synthetic auxins.In one embodiment, the herbicide is glyphosate and the recombinantprotein is a glyphosate-tolerant EPSPS.

A further aspect of the invention includes a method of producing hybridseed including: (a) herbicide application to a transgenic plantincluding a recombinant DNA construct including a protein-codingsequence encoding a recombinant protein that confers herbicide toleranceto the transgenic plant operably linked to a DNA sequence including anmts-siRNA element, wherein the herbicide application is carried outduring the development of the male reproductive tissue of the transgenicplant thereby inducing male-sterility in the transgenic plant; (b)fertilizing the transgenic plant with pollen from a second plant; and(c) harvesting hybrid seed from the transgenic plant. In one embodiment,the transgenic plant is maize. In one embodiment, the herbicide isglyphosate and the recombinant protein is a glyphosate-tolerant EPSPS.In one embodiment, the glyphosate is applied during the development atan effective dose of about 0.125 pounds acid equivalent per acre toabout 8 pounds acid equivalent per acre.

Yet another aspect of the invention includes hybrid seed harvested froma male-sterile transgenic plant that has been fertilized with pollenfrom a second plant, wherein the male-sterile transgenic plant includesa recombinant DNA construct including a protein-coding sequence encodinga recombinant protein that confers herbicide tolerance to the transgenicplant operably linked to a DNA sequence including an mts-siRNA element,and wherein the transgenic plant has been induced to be male-sterile byapplication of an effective amount of herbicide during the developmentof the male reproductive tissue of the transgenic plant. In oneembodiment, the hybrid seed is hybrid transgenic maize seed. In oneembodiment, the herbicide is glyphosate and the recombinant protein is aglyphosate-tolerant EPSPS. In one embodiment, the glyphosate is appliedduring the development at an effective dose of about 0.125 pounds acidequivalent per acre to about 8 pounds acid equivalent per acre. In oneembodiment, the herbicide application prevents at least pollen shed oranther extrusion. In one embodiment, the development of the malereproductive tissue is a stage selected from the group consisting of theV4, V5, V6, V7, V8, V9, V10, V11, V12, V13, and V14 stage of maize plantdevelopment.

EXAMPLES Example 1

This example describes identification of mts-siRNAs and mts-siRNAelements. Bioinformatic analysis of sequencing data from multiple maizesmall RNA libraries identified a group of small RNAs (sRNAs) that wereenriched or specifically expressed in maize tassel. The relativeabundance of these mts-siRNAs in maize tassels ranged from about 50 to631 transcripts per quarter million sequences, which is the normalizedabundance. These sRNAs are identified as siRNAs because of their length(18-26 nucleotides) and their likely origin from a dsRNA precursor.Because of their expression pattern, the male tissue-specific siRNAs arereferred to as “mts-siRNAs”. As used herein, an “expression pattern” isany pattern of differential DNA, RNA, or protein expression. Forexample, a tassel-specific expression pattern refers to specific orenriched expression of a DNA, RNA, or protein in a tassel tissue and/orcell. Examples of the corresponding DNA sequence for mts-siRNAs,referred to herein as “mts-siRNA sequences”, are provided as SEQ ID NO:1-56 and 105-149.

These mts-siRNA sequences were then compared with cDNA sequencecollections. A sequence comparison of the mts-siRNA against a maizeunigene collection (compiled cDNA sequences) using BLAST yielded thesurprising result that a large number of mts-siRNA clustered together,and were even overlapping, within a DNA region found in several closelyrelated, but unique, cDNA sequences. The group of cDNA sequences allcontained such a region, although the DNA sequence of the region varieddue to different combinations and locations of individual mts-siRNAsequences and/or 1-3 nucleotide mismatches to individual mts-siRNAsequences. Such a region defined as having at least one mts-siRNAsequence within a nucleotide sequence window, is referred to herein as a“mts-siRNA element”. In various embodiments, the nucleotide sequencewindow includes at least about 20 contiguous nucleotides (nt) (e.g., atleast 18, 19, 20, 21, 22, 23, or 24 nt), at least about 25 nt, at leastabout 30 nt, at least about 40 nt, at least about 50 nt, at least about100 nt, or at least about 150 nt. Examples of the DNA sequence formts-siRNA elements are provided herein as SEQ ID NO: 57-94 and 96-104.An mts-siRNA element can have more than one mts-siRNA sequence, forexample, at least two, at least three, at least four, at least five, ormore than five mts-siRNA sequences within a given nucleotide sequencewindow. Two or more mts-siRNA sequences within a given mts-siRNA elementmay overlap because at least a portion of their nucleotide sequences areidentical (see Table 5 for examples of mts-siRNAs that have overlappingnucleotide sequences).

Bioinformatic analysis indicated that multiple mts-siRNAs could begenerated from the same RNA transcript, for example a transcriptproduced from one of the cDNA sequences described above as including anmts-siRNA element. Many of the mts-siRNAs were also found to have 1-3mismatches when compared to mts-siRNA elements from across the group ofclosely related cDNA sequences. This is believed to indicate that thesemts-siRNAs are generated from multiple, closely-related transcripts,resulting in a large, closely-related group of mts-siRNAs. Thus, an RNAtranscript produced from a cDNA including an mts-siRNA element(containing multiple mts-siRNA sequences) would be complementary to, andtherefore capable of hybridizing to, multiple mts-siRNAs and/or theircomplements. Thus, a naturally occurring mts-siRNA has an RNA sequencethat is either a perfect or near-perfect complement to an mts-siRNAsequence (e.g., where the mts-siRNA has an RNA sequence with no morethan approximately 1-3 mismatches relative to the mts-siRNA sequence);by extension that same mts-siRNA has an RNA sequence that is a perfector near-perfect complement to a segment of an mts-siRNA element.

A sequence similarity search of the mts-siRNAs against a maize genomicDNA database using BLAST identified multiple loci with significantsimilarity to the mts-siRNA element. These loci were then analyzed foropen reading frames (ORFs), but the identified putative polypeptideswere not found to have significant homology to any known protein.Bioinformatic analysis of the mts-siRNA producing cDNA sequencesindicated that there was no significant sequence homology at thenucleotide level to any known plant gene. These data suggest thatmts-siRNAs could be produced from such loci by processing of dsRNAformed between transcripts of opposite polarity or by processing ofdsRNA from aberrant transcripts due to RNA-dependent RNA polymeraseactivity. It also possible that mts-siRNAs are processed from internalsecondary dsRNA structures that can be formed in some mts-siRNAsproducing transcripts.

Reverse-transcription of the mts-siRNAs provided mts-siRNA sequenceswhich were mapped onto one of the mts-siRNA elements (SEQ ID NO: 87).This is presented in FIG. 1 with the X-axis representing the nucleotideposition from 5′ to 3′ orientation from left to right on the top andfrom right to left on the bottom. The relative abundance of themts-siRNA is given as transcripts per quarter million sequences (tpq)plotted on the Y-axis. As can be seen from FIG. 1, a few mts-siRNAs(circled) are highly represented in the tassel-specific sRNA library(Y-axis). The predicted mts-siRNA sequences are also non-uniformlydistributed across the mts-siRNA element (X-axis).

Example 2

This example illustrates endogenous tassel expression analysis ofmts-siRNAs. The native in planta expression patterns of the mts-siRNAswere analyzed using several different methods. These analyses confirmedthat the sRNAs that hybridize to mts-siRNA elements are enriched inand/or specifically expressed in tassels across maize germplasms (i.e.,the mts-siRNAs are enriched in and/or specifically expressed intassels), and that in an embodiment, an mts-siRNA is enriched in and/orspecifically expressed in the pollen grain at the uninucleate microsporestage of pollen development.

To demonstrate in planta tassel-specific accumulation of the mts-siRNA,three representative mts-siRNA sequences (SEQ ID NO: 26 (1372590), SEQID NO: 8 (648011), SEQ ID NO: 33 (410590)) were used to design probesfor low molecular weight (LMW) northern blot analysis of sRNAs preparedfrom either maize or rice. For these experiments, total RNA wasextracted from plant tissue using TRIzol® reagent (Invitrogen, Carlsbad,Calif.). RNA (7.5 μg) from each sample was denatured at 95° C. for 5minutes before separation on a 17% PAGE gel containing 7 M urea in 0.5×TBE buffer (Allen et al. (2004) Nature Genetics 36:1282-1290). Followingelectrophoresis, the gel was blotted onto a Nytran SuPerCharge® membrane(Whatman-Schleicher & Schuell, Florham Park, N.J.) using Trans-Blot® SDSemi-Dry Electrophoretic Transfer Cell (Bio-Rad, Hercules, Calif.)according to the manufacturer's protocol. The resulting blot wascrosslinked at 1200 microjoules/cm²×100 in a Stratalinker® 1800(Stratagene, Cedar Creek, Tex.). To prepare the probes, an RNA probetemplate was generated by PCR and contained the T7 promoter on one endand one of the small RNA sequences on the opposite end. The sRNAsequences incorporated into the RNA probe template included: [1]Gma-miR159a (miRBase.org accession number MI0001773), which was used asa control for loading; [2] sR1372590 (SEQ ID NO: 26); [3] sR648011 (SEQID NO: 8); and [4] sR410590 (SEQ ID NO: 33). The RNA probes weretranscribed using T7 RNA polymerase, and labelled with digoxigenin (DIG)using the DIG Northern Starter Kit (Roche, Indianapolis, Ind.),according to the manufacturer's protocol. Hybridization was performedwith 100 ng of the DIG-labelled probe in PerfectHyb™ hybridizationbuffer (Sigma, St. Louis, Mo.) at 38° C. for 16 hrs. Detection wasperformed with the DIG Northern Starter Kit according to themanufacturer's protocol, before exposure to Kodak ® Biomax™ XAR film(Sigma, St. Louis, Mo.). The samples tested included all, or a subset ofthe following: maize leaf from plants grown under nitrogen stress; maizeshoot, root or endosperm from plants grown under cold stress; maize leafand root from plants grown under drought stress; maize silk; maize youngtassel; maize mature tassel; unpollinated maize kernels; maize embryo—24days after pollination (DAP); maize kernels—22 DAP; mature maizekernels; maize embryo—mature (dry) kernels; maize endosperm—dry; ricegrain; and rice seedling. The results obtained with the LMW northernanalysis using at least three different mts-siRNA probes (sR1372590,sR648011, and sR410590) showed signal only in the lanes corresponding tothe young tassel and mature tassel lanes, confirming the bioinformaticanalysis and the conclusion that the mts-siRNA expression is highlyenriched in or specific to tassel tissue.

The tissue specificity and accumulation of sRNAs that would recognize anmts-siRNA element was assessed across a wide spectrum of maize germplasmusing LMW northern analysis. For this analysis an mts-siRNA element (SEQID NO: 87, which contains multiple mts-siRNA sequences) was selected.This mts-siRNA element includes the three mts-siRNA sequences used todesign the siRNA probes sR1372590, sR648011, and sR410590, allowingthese probes to be used for the LMW northern analysis of the maizegermplasm samples. For these experiments, RNA was prepared from twentydifferent maize inbred lines with diverse genetic backgrounds, e.g.,with relative maturity rating from 83 to 120 (Table 1). For three ofthese inbred lines (91DUA6, 01DKD2, and LH244), tissue was collectedfrom young tassel, old tassel, leaf, ear, and root. Table 1 provides thecorresponding V-stage and tassel size at collection of young tassel andold tassel. Total RNA was extracted using TRIzol® solution. LMW RNA wasisolated with mirVana™ miRNA isolation kit (cat. no. AM1560, Ambion,Austin, Tex.). LMW northern analysis was done using a Bio-Rad Criterion™Precast 15% TBE-urea acrylamide gel (cat. no. 345-0092, BioRad,Hercules, Calif.). The gel was blotted onto a positive charged membrane(cat. no. 11209272, Roche Applied Systems, Mannheim, Germany). Probeswere labelled with either (1) 32-P-random priming, or (2) with DIG DNAusing Roche PCR labeling kit, or (3) with DIG RNA probe as describedabove. All probes used to probe the northern blots were the reversecomplement to the endogenous transcript or the cDNA sequence of themts-siRNA element. The presence of sRNA that hybridized to thetransgenic mts-siRNA element was specific to tassel; no signal wasdetected for leaf, ear, or root for any of the three inbred maizegenotypes 91DUA6, 01DKD2, and LH244 (FIG. 2).

To determine the temporal expression pattern during tassel developmentof sRNAs which would recognize an mts-siRNA element (SEQ ID NO: 87), LMWnorthern analysis was done. RNA was prepared from young and old tasselfrom different maize inbred lines, see Table 1. The RNA preparation andLMW northern techniques were essentially as described above.

TABLE 1 Inbred germplasm, maturity rating, and tassel development stageyoung tassel old tassel Maturity Tassel size Tassel size Inbred ratingStage (inches) Stage (inches) C3SUD402 108 V9 5 V12 10 HIQA202 113 V9-104.5 V13 13 BEBE788 83 V10-11 10 V13 7.5 BIQA207 103 V10 7 V11 9.5DIDA404 112 V10 2.5-3 V11 9.5 5DA92 107 V10-11 5.7 V12 11 DIDA406 109V10 6.5 V12 9 80DJD5 114 V10 6.5 V11 10 JEDO115 120 V9 2.5 V11-12 10FIDA240 116 V9-10 2.5 V12 11 BIQA347 99 V9-10 3.5 V11-12 10.5-11 HOQA203105 V10-11 5.5 V12-13 11 91DUA6 90 V10-11 10 V12-13 10 BIDA345 95 V10 5V12-13 10 01DKD2 111 V9-10 5 V13 10.5-11 DIDA403 108 V10 2.5-3 V1210.5-11 64DJD1 105 V9-10 2.5-3 V12 10.5-11 DIQA423 108 V9-10 3 V12 9.5BIQA208 102 V10 5.5 V13 9.5 LH244 111 V9-10   1-2.5 V13 10

As seen in FIG. 4, a DIG-labelled RNA probe corresponding to the reversecomplement of an mts-siRNA element (SEQ ID NO: 87) hybridized to sRNA inboth young and old tassel, with the exception of young tassel that was2.5 inches to 3 inches in length: lanes 5 (inbred DIDA404), 9 (inbredJEDO115), 10 (inbred FIDA240), 16 (inbred DIDA403), 17 (inbred 64DJD1),18 (inbred DIQ423), and 20 (inbred LH244). Additionally, this experimentconfirmed no detection of sRNA hybridizing to the mts-siRNA element fromsamples of leaf (lanes 21 and 22) or ear (lanes 23 and 24) from theinbreds BIQA208 and LH244. Collectively these data indicate that thesRNAs that hybridize to the mts-siRNA element are specifically expressedin the tassel from each inbred genotype tested when the tassel isgreater than about 3.5 inches.

In situ hybridization analysis was done to investigate cell specificexpression of an mts-siRNA sequence (sR648011, SEQ ID NO: 8). In maizeanthers, microspores are produced through meiosis and develop intomature pollen. Maize microsporegenesis can be roughly divided into thefollowing stages: meiosis of sporogenous cells, release of tetrads asfree microspores, mitosis of uninucleate microspores to producetricellular pollen, and mature pollen grains. For these experiments,maize tassel before anthesis obtained from maize plants grown understandard conditions in a greenhouse was used. Locked Nucleic Acid (LNA)probes (Integrated DNA Technologies, Coralville, Iowa) were used asindicated below with the position of the LNA indicated by a ‘+’ symbol.The antisense probe was designed to detect the mts-siRNA for sR648011(SEQ ID NO: 8) (5′-Biotin-CAT+GCA+CTG+GTG+AGT+CAC+TGT-3′), while thesense probe was the reverse complement of the antisense probe(5′-Biotin-ACA+GTG+ACT+CAC+CAG+TGC+ATG-3′) for use as a negativecontrol. The LNA probes allow high stringent washes and therefore ensurehighly specific hybridization (Válóczi et al., 2006; Nuovo et al.,2009). All probes were biotin labelled. The samples of maize tassel werefixed in 4% paraformaldehyde in 1×PBS at 4° C. for 36 h, and thendehydrated at 4° C. through a graded ethanol:H₂O series. The tasselswere then placed in 75% EtOH and 25% Histoclear (National Diagnostics,Atlanta, Ga.) for 1.5 h, 50% EtOH and 50% Histoclear for 1.5 h, 25% EtOHand 75% Histoclear for 1.5 h, and 100% Histoclear for 3×1.5 h, all at25° C. Next, the Histoclear was gradually replaced with molten paraplastat 50° C., and the tassels were transferred into molds and stored at 4°C. before sectioning. The paraffin-embedded tassels were sectioned on amicrotome to 8 μm thickness. A series of sections were made from thesame anthers and adjacent sections were then used for probing with thesense or antisense probe, respectively. Prehybridization andhybridization were conducted at 42° C. and washing at 55° C. Detectionof the biotin-labelled LNA probes annealed with the transcripts was witha 1 to 400 dilution of Anti-Biotin-Alkaline Phosphatase (AP) and BMPurple AP Substrate (Roche Applied Science, Indianapolis, Ind.). Imageswere captured from a camera on an Olympus microscope (Center Valley,P.A.). Sections from the same anthers were divided into two groups—onewas used for the antisense probe (FIG. 5, left panel) and the other forthe sense probe (FIG. 5, right panel). The hybridization signal (darkpurple) was detected only on the sections that were hybridized with theantisense probe but not on those that were incubated with the senseprobe (FIG. 5). The strong signal obtained with the antisense probeindicates that this mts-siRNA was abundant (highly expressed) in thepollen grain at the uninucleate microspore stage of pollen development.

Example 3

This example illustrates plant transformation constructs and transgenicplant production. An mts-siRNA element was incorporated into the 3′UTRof a transgene expression cassette and used to produce transgenic maizeplants to test the effect of the element on transgene expression intransgenic plants. An mts-siRNA element (SEQ ID NO: 87) was insertedinto the 3′UTR of a CP4-EPSPS transgene expression cassette for maizetransformation. This mts-siRNA element was selected because it has anabundance of mts-siRNA sequences in it (FIG. 3), including sequences forthree of the siRNA probes (sR1372590, sR648011 and sR410590) used forthe LMW northern analysis in Example 2. This mts-siRNA element alsoallowed testing the effect of mts-siRNA mismatches. The mts-siRNAelement tested here (SEQ ID NO: 87) has a one nucleotide change (CΔT:AAGCTATTGATTCCCTAAGTGCCA) compared to one of the underlying mts-siRNAsequences (SEQ ID NO: 33, used to design Probe sR410590). The mts-siRNAelement was inserted in the transgene cassette in the reverse complementorientation relative to its position in the endogenous cDNA, but theelement is believed to function similarly in both orientations becausetassel-specific siRNA molecules complementary to either strand of themts-siRNA element can be found in maize tassel (FIGS. 1 and 3).

Several CP4-EPSPS/mts-siRNA element expression cassettes wereconstructed (Table 2) and used to transform maize plants. Differentcombinations of expression elements were tested in theCP4-EPSPS/mts-siRNA element expression cassettes. Expression elementssuch as promoters, leaders, introns, chloroplast transit peptides, and3′UTR's needed for efficient and stable expression of a transgene arewell known in the art. The CP4-EPSPS/mts-siRNA element expressioncassettes were designed to include one of two separate promoters;operably linked to a DNA of one of two separate leaders; operably linkedto a DNA of one of two introns; operably linked to one of two DNAmolecules encoding the same chloroplast transit peptide (CTP); operablylinked to a DNA molecule derived from the aroA gene from theAgrobacterium sp. strain CP4 and encoding the CP4-EPSPS protein;operably linked to DNA encoding an mts-siRNA element; operably linked toone of two 3′UTR DNA molecules. Construct 4 contained the wildtypeCP4-EPSPS gene and all the other vectors contained a plant codonoptimized version of the CP4-EPSPS gene. Constructs 3, 5, and 6 (Table2) were designed to determine if an mts-siRNA element incorporated intothe 3′-UTR would produce plants with tassel-specific sensitivity toglyphosate and vegetative glyphosate tolerance. Constructs 4 and 7 arecontrol constructs, lacking an mts-siRNA element.

TABLE 2 Plant transformation constructs Con- Pro- Lead- In- Trans-struct moter er tron CTP gene mts-siRNA 3′UTR 3 A A A A CP4 SEQ ID NO:87 A 4 A A A A CP4 ** A 5 B B B B CP4 SEQ ID NO: 87 A 6 B B B B CP4 SEQID NO: 87 B 7 B B B B CP4 ** A

Transgenic maize plants transformed with one of each of the fiveexpression cassettes were produced using well-known methods. Briefly,maize cells were transformed by Agrobacterium-mediated transformationwith one each of the constructs listed in Table 2 (individually) andregenerated into intact maize plants. Individual plants were selectedfrom the population of plants that showed integrity of the transgeneexpression cassette and resistance to glyphosate. Rooted plants withnormal phenotypic characteristics were selected and transferred to soilfor growth and further assessment. R0 plants were transferred to soilfor growth, sprayed with 0.75 lb/acre glyphosate at V3-V4 followed by0.75 lb/acre glyphosate at V7-V9, and then cross-pollinated with pollenfrom non-transgenic maize plants of the same germplasm (for constructs3, 5, and 6 events) or self-pollinated (for constructs 4 and 7 events)to produce R1 seed. Plants were then selected by a combination ofanalytical techniques, including TaqMan, PCR analysis, and vegetativetolerance to herbicide spray and a reduced (desired) male fertilityrating following herbicide (glyphosate) spray.

Example 4

This example illustrates methods of analyzing transgenic plants in agreenhouse. Transgenic plants transformed with the CP4-EPSPS/mts-siRNAelement expression cassettes were analyzed for vegetative glyphosatetolerance and male fertility. Transgenic plants generated fromconstructs containing the CP4-EPSPS/mts-siRNA element expressioncassettes were found to have vegetative tolerance to glyphosate andinduced male-sterility with late application of glyphosate.

R0 plants were grown in duplicates in the green house and left unsprayedor sprayed with 0.75 lb/acre glyphosate at the (early) V6 stage followedby 0.75 lb/acre glyphosate at the (late) V9 stage. (FIG. 7 and Table 3)The R0 events tested were multi-copy events. All R0 plants that wereunsprayed had normal anther extrusion and fully fertile pollen asdetermined by Alexander staining. All R0 plants that were sprayed hadvegetative tolerance to glyphosate. R0 plants produced from constructs 4and 7, which did not contain the mts-siRNA element, did not show tasselsensitivity to glyphosate or induced male-sterility. R0 plants producedfrom constructs 3, 5, and 6, which did contain the mts-siRNA element,showed tassel sensitivity to glyphosate and induced male-sterility;these plants had no or very few anther extrusions and >99% of the pollenwas non-viable as determined by Alexander staining.

TABLE 3 Glyphosate spray data Early Glyphosate Spray Late GlyphosateSpray Construct Vegetative Tolerance Induced Male-sterility 3 Yes Yes 4Yes No 5 Yes Yes 6 Yes Yes 7 Yes No

These observations demonstrated that the presence of the mts-siRNAelement in the 3′UTR of a transgene cassette led to tassel-specifictransgene silencing of the transgene. Tassel-specific loss of the mRNAtranscript produced by CP4-EPSPS/mts-siRNA element expression cassetteresulted in tassels which were sensitive to glyphosate, producing aplant with induced male-sterility, while the other tissues of the plantwere glyphosate tolerant, producing vegetative glyphosate tolerance andgood female fertility.

Immunolocalization was then used to measure CP4-EPSPS protein in thetransgenic plant tissues. Tassel was obtained from plants transformedwith construct 3 or construct 4 and from non-transgenic maize (LH198).The plants were grown in a greenhouse with 14 hours of light at 80° F.and 8 hours of dark at 70° F. One seed was planted per pot. The potswere randomly arranged on the greenhouse floor. Plants were watered asnecessary and fertilized with 20-20-20 mixture of nitrogen, potassiumand phosphorus, respectively. Plants from construct 3 or construct 4were sprayed with glyphosate at 0.75 lb/acre at the V2 stage to confirmvegetative tolerance to glyphosate. Young tassels were harvested atV10-V11 for anther tissues at microspore mother cell and free microsporestages; mature tassels were harvested at the T7 stage, 1-2 days beforepollen shedding, for anther tissues with fully-developed pollen. Antherswere removed from the tassel spikelet using dissecting forceps andimmediately fixed in 3.7% formaldehyde in phosphate buffered saline(PBS) under gentle vacuum. After washing in PBS, tissues were placed inembedding medium and frozen immediately. Frozen tissue blocks werestored at −80° C. until sectioned in −20° C. microtome and collected onthe charged slides.

Tissue sections were blocked with blocking agent (10% normal goat serum,5% bovine serum albumin, 0.1% Triton X-100 in PBS) for 2 hours. Sectionswere incubated with anti-CP4-EPSPS antibody (1/500 in PBS). Afterwashing the sections three times in PBS, tissue sections were incubatedwith the secondary antibody, goat anti-mouse IgG conjugated with Alexafluorophore 488 (Invitrogen, Eugene, Oreg.). For a negative control,CP4-EPSPS antibody incubation was omitted. As a positive control, anantibody to α-tubulin (Sigma, St. Louis, Mo.), a cytoskeletal proteinexpressed in most cell types, was substituted for the CP4-EPSPS antibodyon separate sections. Both primary and secondary antibodies wereincubated at room temperature for 2-4 hours and then further incubatedovernight at 4° C. After washing, the tissues were imaged with ZeissLaser Scanning Microscope (LSM) 510 META confocal microscope using a 488nm laser for excitation and 500-550 nm for emission filter set. The sameimaging parameter was applied throughout the samples including controls.Fluorescent and bright field images were scanned from each section, andmerged using LSM software afterward to show structural information. Astrong signal was obtained with the anti-CP4-EPSPS antibody in filamenttissue (FIG. 6A, short arrow) and pollen (FIG. 6A, long arrow) in maturetassel from plants generated with construct 4 (FIG. 6A), lacking themts-siRNA element. The plant in FIG. 6A is hemizygous for the transgenecassette, therefore only about 50% of the pollen showed the positiveCP4-EPSPS signal. In contrast, a strong signal was obtained with theanti-CP4-EPSPS antibody only in filament tissue (FIG. 6B, short arrow)and no signal was seen in pollen (FIG. 6B, long arrow) in mature tasselfrom plants generated with construct 3 containing the mts-siRNA element(FIG. 6B). The positive control antibody (anti-alpha-tubulin) showedsignal in pollen within the mature tassel from plants generated fromeither construct 4 or construct 3. The data for the negative controlsshowed the expected absence of signal. The data for the conventionalnon-transgenic control showed the expected absence of signal fromstaining with the anti-CP4-EPSPS antibody and positive signal withstaining with the anti-alpha-tubulin antibody. These data indicate thatno or very few transcripts from the transformation cassette containingthe mts-siRNA element are translated in the pollen, but that thetranscript was translated in the vegetative filament tissue. The loss ofCP4-EPSPS protein expression in pollen correlates to the observedtassel-specific glyphosate sensitivity in plants generated fromconstruct 3.

Example 5

This example illustrates transgenic plant field trial testing for malefertility or sterility. Thirteen confirmed single-copy R1-R3 transgenicplant events generated by transformation with the CP4-EPSPS/mts-siRNAelement expression cassette (construct 3) were tested in field trialsfor efficacy of the expression cassette. In the first year, thirteenevents were tested at one field location. In the second year, eightevents were tested at four field locations. In the third year, fourevents were tested in four field locations. During the three years offield trials, the average male fertility rating (MFR) for eventsgenerated from construct 3 was near or below MFR 2, which is consideredthe industry standard for male-sterility.

The data for one year of efficacy field trials is presented in FIG. 8,with the average MFR produced under three different glyphosate spraytreatment regimens presented in the graph (FIG. 8A) for NK603 (CP4-EPSPStransgenic maize), MON87427 (CP4-EPSPS transgenic maize withglyphosate-inducible male-sterility), and two events from construct 3.Photos of tassel from plants grown during this particular efficacy fieldtrial illustrate fertile tassel when the plants were sprayed withglyphosate at 0.75 lb/acre only at V3 (FIG. 8B); and sterile tassel (noor minimal anther extrusion) on plants sprayed with glyphosate 0.75lb/acre at V3 followed by 0.75 lb/acre at V8 followed by 0.75 lb/acre atV10 (FIG. 8C). For this field trial, the spray regimens were: treatment1 consisted of 0.75 lb/acre glyphosate at V3 (weed control); treatment 2consisted of 0.75 lb/acre glyphosate at V3 (weed control) followed by0.75 lb/acre at V8 followed by 0.75 lb/acre at V10; treatment 3consisted of 0.75 lb/acre glyphosate at V3 (weed control) followed by1.25 lb/acre at V8 followed by 1.25 lb/acre at V10. The later two sprays(i.e., V8 and V10) are referred to as sterility sprays. These resultsindicate that with only weed control glyphosate spray treatment 1 allplants (NK603, MON87427, and construct 3 events) were male fertile. Withglyphosate sterility spray treatment 2, NK603 plants had a MFR=5,MON87427 were sterile with a MFR=2, and events 2 and 3 of construct 3were partially male-fertile with a MFR<3. With glyphosate sterilityspray treatment 3, NK603 plants had a MFR=5, MON87427 were male-sterilewith a MFR<2, and events 2 and 3 of construct 3 were male-sterile with aMFR near or below a score of 2.

Although the average MFR was near or at a score of 2, anther extrusionwas observed in glyphosate treated construct 3 events at S90+3 and S90+6(FIG. 9). For these data, four separate construct 3 events were comparedto MON87427 and NK603 plants for two glyphosate spray regimens:treatment 2 consisted of 1.5 lb/acre glyphosate at V2/V3 (weed control)followed by 0.75 lb/acre glyphosate at growing degree units (GDU) 875(˜V8) followed by 0.75 lb/acre glyphosate at GDU 1025 (˜V10) andtreatment 3 consisted of 1.5 lb/acre glyphosate at V2/V3 (weed control),followed by 1.25 lb/acre glyphosate at GDU 875 (˜V8) followed by 1.25lb/acre glyphosate at GDU 1025 (˜V10). The number of plants per plot(68-74 plants/plot) showing anther extrusion were scored at S90, S90+3and S90+6, where S90 is the day when 90% of the plants in the field areshowing silk; S90+3 is 3 days after S90; and S90+6 is 6 days after S90.As seen in the FIG. 9, at S90 there were 70(±15) NK603 plants per plotshowing anther extrusion for both glyphosate treatment regimens. Incontrast, for the MON87427 and the four construct 3 events, there was1(±12) plant per plot showing anther extrusion at S90 for bothglyphosate treatment regimens. At S90+3 and S90+6, there were 30(±12) to70(±12) plants per plot for the four construct 3 events showing antherextrusion with either glyphosate treatment regimen, nearing that seenfor NK603. The anther extrusion for the MON87427 event remained at theS90 level for both S90+3 and S90+6 time points, and for each glyphosatetreatment regimens. Any plants with >1 extruded anther were scored aspositive for anther extrusion. This late anther extrusion, i.e. S90+3and S90+6, occurs at a time of maize development when there is a maximumgrowth height of the tassel and there is sufficient distance to allowmachine cutting of the tassel with minimal injury to the top two leavesof the maize plant, hence minimal impact on inbred yield. Also, antherextrusion at S90+3 or later is considered to have little impact on seedpurity.

Analysis of pollen viability was conducted to determine if the lowlevel, but consistent anther extrusion observed at S90+3 to S90+6 was anindication of potential late breaking male fertility. FIGS. 10A and 10Billustrate an example of late breaking anther extrusion in tassel from asterility sprayed construct 3 event. The box in FIG. 10A is the portionmagnified in FIG. 10B. An example of late breaking anther extrusion iscircled in FIG. 10B. To determine pollen viability, pollen was gatheredfrom late breaking extruded anther and stained with Alexander stain,FIG. 10C. Pollen was also gathered from non-sprayed construct 3 eventson the same day and stained with Alexander stain as a comparison, FIG.10D. The results of this Alexander staining shows only non-viable pollen(translucent light blue, irregular shape pollen grains) from the latebreaking anthers of sprayed construct 3 events (FIG. 10C). Fully viablepollen appears opaque, dark purple and spherical with Alexander stain(FIG. 10D). In addition to staining pollen collected from individuallyisolated late breaking extruded anther, pollination bags were placed onsome sprayed construct 3 events to determine pollen shed. No noticeablepollen was shed into these pollination bags, which failed to generateany seed when used to cross pollinate recipient ears. This resultsuggested that there was no pollen shed from the late extruding anthersor that any pollen shed is non-viable.

Collectively, these data indicate that although there is low levelanther extrusion from sterility sprayed construct 3 events, theseextruded anthers do not shed viable pollen.

Example 6

This example illustrates transgenic plant field trials testing foryield. Construct 3 R2 plants were tested for inbred and hybrid yield.For inbred yield, construct 3 R2 plants were tested in four fieldlocations for yield, vegetative tolerance to glyphosate spray, andmale-sterility with glyphosate spray. For these field trials, fourevents from construct 3 were planted in plots of 68-74 plants/plot. Thespray treatments were: treatment 1 consisted of 1.5 lb/acre glyphosateat V3 (weed control); treatment 2 consisted of 1.5 lb/acre glyphosate atV3 followed by 0.75 lb/acre at V8 followed by 0.75 lb/acre at V11;treatment 3 consisted of 1.5 lb/acre glyphosate at V3 followed by 1.25lb/acre at V8 followed by 1.25 lb/acre at V11. As can be seen in FIG.11, the construct 3 events at all three glyphosate treatment regimensshowed good vegetative tolerance (white bars) as measured by plantheight and good inbred yield (black bars) as measured as bushels(Bu)/acre. These same events were fully male-fertile when treated withonly the weed control glyphosate treatment regimen (treatment 1), butwere male-sterile with a MFR score of equal to or less than 2 (graybars) when treated with glyphosate treatment regimens 2 or 3. Thehorizontal bar on FIG. 11 indicates the industry standard for sterility,MFR 2. NK603 is provided for comparison. The measures of yield of inbredgrain for construct 3 events and NK603 with glyphosate spray areprovided in Table 4, where MST=% moisture of the grain, TWT=test weight(a density rating, typically pounds per bushel), and S50D is number ofdays to 50% silking of the ears in the plot. There was no significantdifference (nd) measured in yield for any of the four construct 3 eventstested with either glyphosate treatment 2 or 3 compared to the NK603control.

TABLE 4 Inbred grain yield measures Comparison of Treatments 2 and 3 toTreatment 1 Events MST TWT S50D cNK603 nd nd nd Event 1 nd nd nd Event 2nd nd nd Event 4 nd nd nd Event 3 nd nd nd

For F1 hybrid grain yield, construct 3 R3 events were tested in fourfield locations. For these hybrid yield field trials, a non-transgenicfemale inbred (Null), line MON87427, and three events from construct 3,all in the same genetic background, were cross pollinated with a maleMON810/MON88017 tester to generate F1 hybrid seed. The F1 hybrid seedgenerated from each of these crosses was planted in standard plots of68-74 plants/plot. The spray treatments consisted of treatment 1 of noglyphosate spray; treatment 2 of 2.25 lb/acre glyphosate at V4 followedby 2.25 lb/acre at V7; treatment 3 of 2.25 lb/acre glyphosate at V4followed by 2.25 lb/acre at V7 followed by 2.25 lb/acre at V10. The F1plants were open pollinated to generate F2 grain, which is the yieldmeasured in bushels/acre (Bu/acre). All three construct 3 events showedequivalent F1 hybrid grain yield at all glyphosate treatment regimenswhen compared to the control crosses of NullxMON810/MON88017 andMON87427xMON810/MON88017 (FIG. 12).

Example 7

This example illustrates male fertility restoration in F1 hybrid plants.F1 hybrid plants generated from a cross of construct 3 events as thefemale parent were tested for male fertility. Three different F1 hybridcrosses were set-up: non-transgenic female x MON88017 male; MON87427female x MON88017 male; and construct 3 event female x MON88017 male.The F1 hybrid seed was harvested from each of the three crosses, plantedin a field, and sprayed with glyphosate at 1.125 lb/acre at V4 followedby 1.125 lb/acre at V10. Male fertility in F1 was assessed by malefertility rating (MFR) and by Alexander viability staining of thepollen. For each of the crosses, the MFR of the F1 hybrid plants was 5,or fully fertile. The Alexander viability staining showed 50% of thepollen produced by the F1 hybrid of each of the crosses was viable, asexpected. (FIG. 13) These data indicate that male fertility can befunctionally restored in F1 hybrid plants produced fromglyphosate-inducible male-sterile transgenic plants transformed with aCP4-EPSPS/mts-siRNA element expression cassette.

Example 8

This example illustrates variant and chimeric mts-siRNA elementconstruction. Individual mts-siRNA were mapped onto an mts-siRNA elementas presented in FIG. 3; the X-axis indicates the nucleotide positionfrom 5′ to 3′ orientation from left to right on the top and from rightto left on the bottom, and the Y-axis indicates the relative abundanceof the mts-siRNA is indicated as transcripts per quarter millionsequences (tpq). The mts-siRNAs were also non-uniformly distributedacross the mts-siRNA element (X-axis).

Using this information, variants of an mts-siRNA element and/or chimerasproduced using one or more mts-siRNA element(s) were engineered tocontain more (or fewer) total mts-siRNA sequences (optionally oralternatively, one or more mts-siRNA sequence(s) is added or deleted),resulting in more (or less) silencing of an operably linkedprotein-coding sequence. Such variants or chimeric mts-siRNA elementsare useful for increasing or decreasing the selective suppression of theexpression of a recombinant protein in a male reproductive tissue of atransgenic plant.

Examples of variants and chimeras of mts-siRNA elements were constructedusing fragments of SEQ ID NO: 87. The first variant (SEQ ID NO: 88) wasconstructed using a 104 nucleotide fragment from the 5′-end of SEQ IDNO: 87. The second variant (SEQ ID NO: 89) was constructed using an 80nucleotide fragment from the 3′-half of SEQ ID NO: 87. Chimericmts-siRNA elements were constructed by joining one fragment (SEQ ID NO:88) to another fragment (SEQ ID NO: 89) to form new chimeric mts-siRNAelements (SEQ ID NO: 90 and SEQ ID NO: 91). Additional chimericmts-siRNA elements were constructed by joining three individualmts-siRNA contained within SEQ ID NO: 87: a first chimera (SEQ ID NO:92) was constructed by joining mts-siRNA sequences SEQ ID NO: 26, 27,and 8; a second chimera (SEQ ID NO: 93) was constructed by joiningmts-siRNA sequences SEQ ID NO: 10, 33, and 5; a third chimera (SEQ IDNO: 94) was constructed by joining mts-siRNA sequences SEQ ID NO: 26,10, and 33. These variants and chimeras can be operably linked toprotein-coding sequences to produce recombinant DNA constructs (see FIG.14) that can be tested in plants and plant cells for selectivesuppression of a recombinant protein encoded by the protein-codingsequence in a male reproductive tissue of a transgenic plant.

Example 9

This example illustrates design of variant and chimeric mts-siRNAelements. Variant and chimeric mts-siRNA elements were designed based ona 300-nucleotide (nt) long mts-siRNA element having SEQ ID NO: 81, whichis similar to the 300-nucleotide mts-siRNA elements having SEQ ID NO: 82and 87. A highly conserved consensus sequence for mts-siRNA elements SEQID NO: 81, 82, and 87 is provided by SEQ ID NO: 96. Individually, eachof these are also useful as an mts-siRNA element or as the basis ofdesigning variant or chimeric mts-siRNA elements, e.g., by selectingfragments of an mts-siRNA element identified from genomic sequence orcDNAs, such as fragments including at least one mts-siRNA sequence, andcombining or concatenating such fragments.

Two fragments within SEQ ID NO: 81 were selected; fragment A (SEQ ID NO:97) contained 104 contiguous nucleotides from the 5′ region (positions1-104) of SEQ ID NO: 81 and fragment B (SEQ ID NO: 98) contained 80contiguous nucleotides from the 3′ region (positions 215-294) of SEQ IDNO: 81; it is clear that either fragment A (SEQ ID NO: 97) or fragment B(SEQ ID NO: 98) individually are mts-siRNA elements containing at leastone mts-siRNA sequence. The location of fragments A and B (indicated byunderlined text) is shown in the following full sequence of SEQ ID NO:81, which also indicates the location of mts-siRNA sequences (indicatedby italicized text; italicized segments of greater than 18 contiguousnucleotides can include more than one overlapping mts-siRNA sequences)found to map to this mts-siRNA element:

(SEQ ID NO: 81) GGACAACAAGCACCTTCTTGCCTTGCAAGGCCTCCCTTCCCTATGGTAGCCACTTGAGTGGATGACTTCACCTTAAAGCTATCGATTCCCTAAGTGCCAGACATAATAGGCTATACATTCTCTCTGGTGGCAACAATGAGTCATTTTGGTTGGTGTGGTAGTCTATTATTGAGTTTGTTTTGGCACCGTACTCCCATGGAGAGTACAAGACAAACTCTTCACCGTTGTAGTCGTTGATGGTATTGGTGGT GACGACATCC TTGGTGTGCATGCACTGGTGAGTCACTGTTGTACTCGGCG.Variant mts-siRNA elements were designed using the “A” and “B”fragments, including an “A+B” mts-siRNA element (SEQ ID NO: 99) and a“B+A” mts-siRNA element (SEQ ID NO: 100). A chimeric element (SEQ ID NO:101) was designed to include the mts- mts-siRNA sequences (shown abovein italicized text in SEQ ID NO: 81) that were found to map to themts-siRNA element (SEQ ID NO: 81).

Similarly, a 251-nt long mts-siRNA element (SEQ ID NO: 102) and a 121-ntlong mts-siRNA element (SEQ ID NO: 103, a fragment of SEQ ID NO: 102,i.e., the contiguous segment located at nucleotide positions 47-167 ofSEQ ID NO: 102) were identified from maize genomic sequence(Zm_B73_CR10::Segment{75361491 . . . 753617421}) as tassel-specific andcorresponding to mts-siRNAs from young tassel (maize LH244, library 347;individually identified mts-siRNAs in some cases overlap over much oftheir sequence and vary by only a few nucleotides; see Table 5). Basedon SEQ ID NO: 102 and 103 a chimeric mts-siRNA element (SEQ ID NO: 104)was designed.

TABLE 5 mts-siRNAs mapped to SEQ ID NO: 103 expression sRNA ID (tpq for(specific lib9, raw SEQ library to each map map counts for ID SequenceID library) start* end* strand others) NO: (as DNA equivalent) 347 710618  48  72 -1 1 105 ACCAAAGCCGCAATACTTAGCCCTA 347     325  49  72-1 667 106 ACCAAAGCCGCAATACTTAGCCCT 9   75221  49  72 -1 14.0375 107ACCAAAGCCGCAATACTTAGCCCT 9   79587  49  70 -1 1.7547 108CAAAGCCGCAATACTTAGCCCT 347 1443964  49  70 -1 1 109CAAAGCCGCAATACTTAGCCCT 347 1798947  50  72 -1 1 110ACCAAAGCCGCAATACTTAGCCC 9  993198  51  74  1 5.264 111GGCTAAGTATTGCGGCTTTGGTAG 346 2511625  56  79 -1 1 112GACAACTACCAAAGCCGCAATACT 347 1978935  62  84 -1 1 113GATATGACAACTACCAAAGCCGC 347  955660  63  86 -1 1 114TAGATATGACAACTACCAAAGCCG 347 1183103  64  84 -1 1 115GATATGACAACTACCAAAGCC 347   36752  73  96 -1 12 116ATCAAAAGTTTAGATATGACAACT 347  151532  75  98  1 4 117TTGTCATATCTAAACTTTTGATAG 347    1372  97 120 -1 197 118ACGAGTACTCTAACATATAAGACT 347  316040  97 117 -1 2 119AGTACTCTAACATATAAGACT 347 1310155  98 121  1 1 120GTCTTATATGTTAGAGTACTCGTT 347   26503  99 122  1 15 121TCTTATATGTTAGAGTACTCGTTA 347  490490 109 132 -1 2 122ATCAAAACCCTAACGAGTACTCTA 347 1125767 114 137 -1 1 123AGACAATCAAAACCCTAACGAGTA 347  442804 115 138 -1 2 124GAGACAATCAAAACCCTAACGAGT 347  965720 118 141 -1 1 125CAGGAGACAATCAAAACCCTAACG 345 1549424 119 142 -1 1 126ACAGGAGACAATCAAAACCCTAAC 347  311196 120 143 -1 2 127CACAGGAGACAATCAAAACCCTAA 347     190 121 144 -1 1018 128ACACAGGAGACAATCAAAACCCTA 346  591709 121 144 -1 2 129ACACAGGAGACAATCAAAACCCTA 347  363241 121 143 -1 2 130CACAGGAGACAATCAAAACCCTA 347 1603891 121 141 -1 1 131CAGGAGACAATCAAAACCCTA 347  135176 122 144 -1 4 132ACACAGGAGACAATCAAAACCCT 347   48157 123 146  1 9 133GGGTTTTGATTGTCTCCTGTGTAT 347 1866298 123 144 -1 1 134ACACAGGAGACAATCAAAACCC 347 1707358 124 147 -1 1 135AATACACAGGAGACAATCAAAACC 347 1788406 129 146  1 1 136 TGATTGTCTCCTGTGTAT347  519539 130 153  1 2 137 GATTGTCTCCTGTGTATTTACCCT 347  383791 133156 -1 2 138 GAGAGGGTAAATACACAGGAGACA 347  273115 135 158  1 2 139TCTCCTGTGTATTTACCCTCTCGC 345 1244664 135 157 -1 1 140CGAGAGGGTAAATACACAGGAGA 346 1460995 135 157 -1 1 141CGAGAGGGTAAATACACAGGAGA 347  697148 135 157  1 1 142TCTCCTGTGTATTTACCCTCTCG 347 1716970 136 159  1 1 143CTCCTGTGTATTTACCCTCTCGCA 347  839648 137 157 -1 1 144CGAGAGGGTAAATACACAGGA 347    8578 145 168 -1 38 145TACAATAAGTGCGAGAGGGTAAAT 9  519321 145 168 -1 8.7734 146TACAATAAGTGCGAGAGGGTAAAT 347  423280 145 167 -1 2 147ACAATAAGTGCGAGAGGGTAAAT 347  377787 146 168 -1 2 148TACAATAAGTGCGAGAGGGTAAA 9  444803 146 167 -1 1.7547 149ACAATAAGTGCGAGAGGGTAAA *nucleotide position within SEQ ID NO: 103

Example 10

This example illustrates vectors and transgenic plant cells, tissues,and plants containing recombinant DNA constructs including aprotein-coding sequence encoding a recombinant protein and an mts-siRNAelement operably linked to the protein-coding sequence.

A plant transformation vector comprising a recombinant DNA construct isused for Agrobacterium-mediated transformation of maize cells. Thistransformation vector includes DNA for Agrobacterium-mediated transferof T-DNA, an expression cassette (promoter operably linked to a DNAsequence of interest), a selectable marker expression cassette (forconvenient selection of the transformed maize cells or plants), and DNAfor maintenance of the vector in E. coli (e.g., an E. coli origin ofreplication sequence). In one embodiment, the transformation vectorincludes an expression cassette comprising a recombinant DNA constructflanked by right and left border sequences from Agrobacterium, whereinthe recombinant DNA construct includes the herbicide tolerance transgeneCR-AGRtu.aroA-CP4.nat (provided as SEQ ID NO: 95) as the DNA sequenceencoding a recombinant protein. The herbicide tolerance transgeneCR-AGRtu.aroA-CP4.nat is operably linked to the mt-siRNA provided as SEQID NO: 81 as the DNA sequence encoding an mts-siRNA element.

Transformation vectors for expressing different recombinant DNAconstructs are constructed by inserting a polynucleotide including anmts-siRNA element (e.g., SEQ ID NO: 57-94 or 97-104) into the planttransformation vector. The mts-siRNA element is inserted adjacent to theDNA sequence encoding a recombinant protein or within the 3′untranslated region of the DNA sequence encoding a recombinant protein.Such plant transformation vectors are useful for making transgenicplants that can be induced to be male-sterile by the application ofherbicide.

Methods for transformation of plants are well-known in the art. Forexample, maize plants of a transformable line are grown in thegreenhouse and ears are harvested when the embryos are 1.5 to 2.0 mm inlength. Ears are surface sterilized with 80% ethanol, followed by airdrying. Immature embryos are isolated from individual kernels fromsterilized ears. Prior to inoculation of maize cells, individualcultures of Agrobacterium each containing a transformation vector forexpressing at least one of the recombinant DNA constructs of thisinvention are grown overnight at room temperature. Immature maize embryocell cultures are inoculated with Agrobacterium, incubated at roomtemperature with Agrobacterium for 5 to 20 minutes, co-cultured withAgrobacterium for 1 to 3 days at 23 degrees Celsius in the dark,transferred to a selection medium and cultured for approximately 2 weeksto allow embryogenic callus to develop. Embryogenic callus istransferred to a culture medium containing 100 mg/L paromomycin andsubcultured at about two week intervals. Multiple events of transformedplant cells are recovered 6 to 8 weeks after initiation of selection.

Transgenic maize plants are regenerated from transgenic plant cellcallus for each of the multiple transgenic events resulting fromtransformation and selection, by placing transgenic callus of each eventon a medium to initiate shoot and root development into plantlets whichare transferred to potting soil for initial growth in a growth chamberat 26 degrees Celsius, followed by growth on a mist bench beforetransplanting to pots where plants are grown to maturity. Theregenerated plants are self-fertilized. First generation (“R1”) seed isharvested. Plants grown from the R1 seed (“R2” plants) are used toproduce progeny.

Example 11

This example illustrates methods of selecting mts-siRNA sequences andmts-siRNA elements for use in recombinant DNA constructs including aprotein-coding sequence encoding a recombinant protein and an mts-siRNAelement operably linked to the protein-coding sequence.

One method of verifying efficacy of an mts-siRNA element for selectivelysuppressing the expression of a recombinant protein in a malereproductive tissue of a transgenic plant involves use of a protoplastassay wherein plant cell protoplasts are co-transformed with: (a) avector containing a recombinant DNA construct including a protein-codingsequence and a mts-siRNA element operably linked to the protein-codingsequence; and (b) RNA(s) having the sequence of the siRNA(s)corresponding to the mts-siRNA element(s) (or alternatively, themts-siRNA sequence(s)), wherein the level of expression of therecombinant protein is expected to be inversely proportional to thedegree to which the mts-siRNA element is cleaved by the RNA(s).

This is illustrated by the following non-limiting example. The assay wascarried out on two mts-siRNA sequences (corresponding to two siRNAsfound to be highly expressed in maize tassel). In brief, maize leafprotoplasts were co-transformed with: (a) a plasmid (3micrograms/320,000 cells) containing a recombinant DNA constructincluding a protein-coding sequence encoding a recombinant protein(CP4-EPSPS, SEQ ID NO: 95) and an mts-siRNA element (SEQ ID NO: 81), and(b) a first dsRNA with a first strand having the sequence SEQ ID NO: 150in 5′ to 3′ direction and a second strand being the complement of thefirst, and a second dsRNA with a first strand having the sequence SEQ IDNO: 151 in 5′ to 3′ direction and a second strand being the complementof the first. The dsRNAs (from Integrated DNA Technologies, Inc.,Coralville, Iowa) were tested at 0, 5, 25, or 50 nanograms/320,000cells, with the total RNA used in each co-transformation assay adjustedwith “filler” RNA consisting of either miRNA395 (as the mature 21-mer,provided as dsRNA) or yeast tRNA to 50 nanograms/320,000 cells. Thelevel of CP4-EPSPS protein was determined by ELISA and used to evaluatethe ability of the tested dsRNAs to suppress expression of therecombinant protein. Results are provided in Table 6.

TABLE 6 Level of CP4-EPSPS protein CP4-EPSPS protein (ng/mg dsRNA testeddsRNA (ng) filler RNA (ng) total protein) none (control) 0 50 317  SEQID NO: 150 5 45 294  25 25 167* 50 0 114* SEQ ID NO: 151 5 45 315  25 25223* 50 0  91* *statistically significant from control

Each of the dsRNAs (SEQ ID NO: 150 and 151) strongly suppressedCP4-EPSPS expression (indicated by decreased CP4-EPSPS proteinaccumulation) when co-transformed with the plasmid containing therecombinant DNA construct including the CP4-EPSPS protein-codingsequence and mts-siRNA element. The observed suppression of CP4-EPSPSwas dose-dependent on the amount of dsRNAs and independent of the typeof filler RNA. Suppression of CP4-EPSPS was not observed in controlsamples co-transformed with filler RNA in place of the test dsRNAs.

Example 12

This example illustrates recombinant DNA constructs, vectors, andtransformed plants of the invention. Vectors and transformation methodssimilar to those described in Example 10 were used to produce stablytransformed maize plants containing in their genome a recombinant DNAconstruct including a protein-coding sequence operably linked to a DNAsequence comprising an mts-siRNA element. Six combinations of constructdesign/mts-siRNA element were tested (see Table 7). Plants were sprayedtwice (at V5 and V8) with 0.75 lb ae/A Roundup WeatherMAX®. Results areprovided in Table 7. For each construct design/mts-siRNA elementcombination, about 20 plants were left unsprayed for comparison toglyphosate-sprayed plants. Unsprayed plants all shed pollen and had goodmale fertility (data not shown). The maize plants transformed withconstruct design B exhibited more pronounced male-sterility than themaize plants transformed with construct design A. Construct designs (5′to 3′, left to right) were Construct A is promoter A/intron A/transitpeptide A/CP4-EPSPS (SEQ ID NO: 95)/mts-siRNA element/3′UTR andConstruct B is promoter B/intron B/transit peptide B/CP4-EPSPS/mts-siRNAelement/3′UTR. As used below, “n.m.” means not measured. The malefertility rating (MFR) scale is: 5=anther emergence is normal, pollenvolume is the same as unsprayed plots but may or may not shed pollen;4=anther emergence 50% of normal, but are shedding slightly or notshedding normal amounts of pollen; 3=tassel looks normal but there issporadic anther extrusion (>10 anthers per tassel) and little or nopollen being shed; 2.5=no pollen shed, anthesis is greatly reduced (<10anthers per tassel) or is very late (1 week) relative to the end ofsilking; 2=no pollen shed, no anthesis or anthesis is very late (1 week)relative to end of silking; and 1=no pollen shed, tassel has abnormalstick phenotype or anthesis is delayed two or more weeks after silking.S90 is when 90% plants have silk ready for pollination and S90+3 is 3days after S90.

TABLE 7 Construct design/mts-siRNA element spray data mts-siRNA PollenMFR at % Construct element Vegetative shedding S90 to abnormal design*SEQ ID NO: damage at S90 S90 + 2 pollen A 97 No No 5 100% A 97 No No 2.5100% A 97 No No 2 100% A 98 No Yes 5 n.m. A 98 No No 3  60% A 98 No No 5 70% A 98 No No 2.5 100% A 104 No No 4 n.m. A 104 No No 2  50% A 104 NoNo 2.5 100% B 101 No No 2 n.m. B 101 No No 2.5 100% B 101 No No 4 n.m. B101 No No 2 n.m. B 101 No No 2.5 n.m. B 101 No No 2.5 100% B 101 No No 2100% B 101 No No 2.5 100% B 101 No No 2.2 n.m. B 101 No No 3  20% B 101No No 2 100% B 97 No No 2.5 n.m. B 97 No No 2 100% B 97 No No 2.5 100% B97 No No 4 100% B 97 No No 2 n.m. B 98 No No 2 100% B 98 No No 2.5 100%B 98 No No 2 n.m. B 98 No No 2.5 n.m.

All of the materials and methods disclosed and claimed herein can bemade and used without undue experimentation as instructed by the abovedisclosure. The above examples are included to demonstrate embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples represent techniquesdiscovered by the inventor to function well in the practice of theinvention, and thus can be considered to constitute preferred modes forits practice. However, those of skill in the art should, in light of thepresent disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

What is claimed is:
 1. A recombinant DNA construct comprising aprotein-coding sequence operably linked to a DNA sequence comprising anmts-siRNA element.
 2. The recombinant DNA construct of claim 1, whereinsaid mts-siRNA element comprises at least one mts-siRNA sequence.
 3. Therecombinant DNA construct of claim 2, wherein said mts-siRNA sequence isselected from the group consisting of SEQ ID NO: 1-56 and 105-149. 4.The recombinant DNA construct of claim 1, wherein said mts-siRNA elementis selected from the group consisting of SEQ ID NO: 57-94 and 96-104. 5.The recombinant DNA construct of claim 1, wherein the expression of saidprotein-coding sequence in a transgenic plant confers at leastvegetative herbicide tolerance to said plant.
 6. The recombinant DNAconstruct of claim 5, wherein said protein-coding sequence encodes aglyphosate-tolerant EPSPS.
 7. A method of making a recombinant DNAconstruct comprising identifying an mts-siRNA element comprising atleast one mts-siRNA sequence and operably linking said mts-siRNA elementto a protein-coding sequence.
 8. The method of claim 7, wherein said atleast one mts-siRNA sequence is at least one selected from the groupconsisting of SEQ ID NO: 1-56 and 105-149 or said mts-siRNA element isselected from the group consisting of SEQ ID NO: 57-94 and 96-104. 9.The method of claim 7, wherein said mts-siRNA element istassel-specific.
 10. A transgenic plant having in its genome therecombinant DNA construct of claim
 1. 11. A seed, progeny, or plant partof the transgenic plant of claim
 10. 12. The transgenic plant of claim10, wherein said transgenic plant is a monocotyledonous plant.
 13. Thetransgenic plant of claim 10, wherein said transgenic plant is a maizeplant.
 14. A method of selectively suppressing the expression of arecombinant protein in a male reproductive tissue of a transgenic plantcomprising expressing in said transgenic plant a recombinant DNAconstruct of claim
 1. 15. The method of claim 14, wherein said malereproductive tissue is a tassel of a maize plant.
 16. The method ofclaim 14, wherein said mts-siRNA element comprises at least threemts-siRNA sequences.
 17. The method of claim 14, wherein said mts-siRNAelement comprises at least one mts-siRNA sequence selected from thegroup consisting of SEQ ID NO: 1-56 and 105-149.
 18. The method of claim14, wherein said wherein said mts-siRNA element is selected from thegroup consisting of SEQ ID NO: 57-94 and 96-104.
 19. The method of claim14, wherein the expression of said recombinant protein in a transgenicplant confers at least vegetative herbicide tolerance to said plant. 20.The method of claim 19, wherein said recombinant protein is aglyphosate-tolerant EPSPS.
 21. A method of inducing male-sterility in atransgenic plant comprising applying an effective amount of an herbicideto a transgenic plant comprising a recombinant DNA construct of claim 1,wherein said herbicide application is carried out during the developmentof the male reproductive tissue of said transgenic plant therebyinducing male-sterility in said transgenic plant.
 22. The method ofclaim 21, wherein said transgenic plant is a maize plant.
 23. The methodof claim 21, wherein said herbicide application prevents at least pollenshed or anther extrusion.
 24. The method of claim 21, wherein saiddevelopment of the male reproductive tissue is a stage selected from thegroup consisting of the V4, V5, V6, V7, V8, V9, V10, V11, V12, V13, andV14 stage of maize plant development.
 25. The method of claim 21,wherein said herbicide is selected from the group consisting of acetylcoenzyme A carboxylase (ACCase) inhibitors, acetolactate synthase (ALS)inhibitors, photosystem II (PSII) inhibitors, protoporphyrinogen oxidase(PPO) inhibitors, 4-hydroxyphenyl pyruvate dioxygenase (HPPD)inhibitors, 5-enolypyruvyl shikimate 3-phosphate synthase (EPSPS)inhibitors, glutamine synthetase (GS) inhibitors, and synthetic auxins.26. The method of claim 21, wherein said herbicide is glyphosate andsaid recombinant protein is a glyphosate-tolerant EPSPS.
 27. A method ofproducing hybrid seed comprising: a. applying an effective amount ofherbicide to a transgenic plant comprising a recombinant DNA constructof claim 1, wherein said herbicide application is carried out during thedevelopment of the male reproductive tissue of said transgenic plantthereby inducing male-sterility in said transgenic plant; b. fertilizingsaid transgenic plant with pollen from a second plant; and c. harvestinghybrid seed from said transgenic plant.
 28. The method of claim 27,wherein said transgenic plant is maize.
 29. The method of claim 27,wherein said herbicide is glyphosate and said recombinant protein is aglyphosate-tolerant EPSPS.
 30. The method of claim 29, wherein saidglyphosate is applied during said development at an effective dose ofabout 0.125 pounds acid equivalent per acre to about 8 pounds acidequivalent per acre.
 31. Hybrid seed harvested from a male-steriletransgenic plant that has been fertilized with pollen from a secondplant, wherein said male-sterile transgenic plant comprises arecombinant DNA construct of claim 1 and has been induced to bemale-sterile by application of an effective amount of herbicide duringthe development of the male reproductive tissue of said transgenicplant.